Gonadotropin releasing hormone (GnRH) neurons originate in or near the olfactory placode and migrate into the forebrain during embryogenesis. Disruption of GnRH neuronal migration or production results in abnormal reproductive function in humans. Axl is a member of the TAM family of receptor tyrosine kinases (Tyro3, Axl and Mer) whose major ligand is growth arrest specific gene 6 (Gas6). Gas6 activation of Axl and Tyro3 promotes GnRH neuronal cell migration and survival. Axl/Tyro3 null mice demonstrate delayed first estrus, abnormal estrus cyclicity and prolonged proestrus. During embryogenesis, there is a decreased number and mistargeting of immunoreactive GnRH neurons, confirming the central role of Axl and Tyro3 in GnRH neuronal development and function. We show new preliminary data that human subjects with Kallmann Syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (nIHH) harbor mutations in Axl, supporting the importance of understanding the role of the TAM system in reproduction. In this application: Aim 1 will determine the ligand dependence of the TAM family effects on reproduction and GnRH neuron survival and migration in vivo. Comparison of Axl/Tyro3 null and Gas6 null mice will define the relative importance of the TAM member and/or ligand during both early GnRH neuron development and their impact on the timing of sexual maturation and reproductive competence. Aim 2 will delineate the neuronal-specific signaling pathways by which Axl or Tyro3 promote GnRH neuronal survival and migration. Studies in GnRH neuronal cells outlined the importance of PI3K/Akt and ERK signaling for survival, and of p38MAPK for migration downstream of Axl and Tyro3. An ex vivo model of embryonic brain slices from control, Axl/Tyro3 null and Gas6 null mice in the presence or absence of specific inhibitors of PI3K, ERK and/or p38MAPK will dissect the role of each in neuronal survival and migration. Oligonucleotide microarrays will identify the neuronal specific components downstream of TAM signaling that promote GnRH survival and migration, which then will be confirmed in the ex vivo brain slice model. Aim 3 will define the functional significance of novel TAM mutations detected in KS and nIHH subjects. We have identified the first AXL mutations in subjects with KS and nIHH, not found in SNP databases or community controls. Functional analysis of the effects of these mutations on TAM signaling and survival and migration in GnRH neuronal cells will be performed. Screening for mutations in other TAM family members and testing the functional impact of these changes will clarify the range of human TAM mutations leading to reproductive disorders. Mutations in components of TAM signaling pathways may contribute to reproductive disorders in humans, justifying the importance of further dissection of the function of this novel system during GnRH neuron development. PUBLIC HEALTH RELEVANCE: Our goals are to identify and characterize novel factors involved in Gonadotropin releasing hormone (GnRH) neuron development, a process that is required for normal reproductive function in mice and humans. Studies will define the roles of Axl and Tyro3 receptor tyrosine kinases and their common ligand, growth arrest specific gene-6 (Gas6), in GnRH neuron migration and survival during embryogenesis and postnatally. Exciting new data have identified novel AXL mutations in patients with Kalmann Syndrome and normosmic idiopathic hypogonadotropic hypogonadism, suggesting the importance of this system in human reproductive biology and justifying further analysis of this system to expand our understanding of GnRH neuron development and function.